DESCRIPTION: (Applicant's Abstract) We plan to evaluate the role of cdk5-catalyzed phosphorylation of DARPP-32 at Thr75, and the consequent regulation of PKA, in mediating the actions of cocaine and amphetamine. Addiction involves profound changes in dopaminergic neurotransmission in the brain. DARPP-32, a dopamine-regulated phosphoprotein enriched in medium spiny neurons plays an obligatory role in mediating the actions of dopamine. DARPP-32 is now recognized to be a dual function protein. DARPP-32 is an inhibitor of protein phosphatase 1 when phosphorylated at Thr34 by PKA and an inhibitor of PKA when phosphorylated at Thr75 by cdkS, a member of the cyclin-dependent kinase family. Chronic cocaine treatment up-regulates the cdk5 gene and increases levels of phospho-Thr75-DARPP-32 in the brain. Moreover, inhibitors of cdk5 potentiate the locomotor-sensitizing effects of cocaine in rats. Regulation of DARPP-32 by cdk5 represents a significant and heretofore-unknown mechanism of dopamine signaling and psychostimulant action in the brain. We propose to analyze the role of cdk5 signaling in the actions of cocaine by examining the effects of cdk5 inhibitors on the biochemistry, physiology, and behavior of rats given chronic cocaine (Aim I). These studies will also employ gene-knockout mice to identify the contributions of selected elements of the cdk5/DARPP-32 signaling cascade to psychostimulant effects. Specifically, we will examine the role of 1) cdk5, 2) Thr75 of DARPP-32, and 3) Ser845 of the AMPA receptor subunit, GluRl, a substrate for phospho-Thr75 DARPP-32, in mediating effects of psycho-stimulants on brain biochemistry, cellular physiology, gene expression, and behavior. These effects include regulation of: 1) gene expression using in situ hybridization and gene chip techniques, with the affected gene products being further analyzed with regard to their distribution, phosphorylation and possible interactions with other components of the dopamine signaling cascade; 2) locomotor activity, place-preference conditioning, drug self-administration and escalation of drug intake; and 3) neurotransmitter receptor phosphorylation. For these studies we are engineering mice expressing a conditional, tissue-specific knockout of the cdk5 gene (Aim II), mice expressing DARPP-32 with a non-phosphorylatable Thr75 (Thr75-A1a mutants)(Aim III), and mice expressing GluRl, with a Ser845-A1a mutation (Aim IV). These studies should contribute to our understanding of the molecular basis for the development and expression of psychostimulant actions leading to drug addiction.